Recent evidence emphasizing the complexity of amino acid pools in mammalian tissues has left no clear basis for interpreting isotopic data on protein turnover. We propose to define precursor amino acid pools in muscle which either are or can be used under specific circumstances as the immediate precursor for protein, in order to elucidate the specific loci of the anabolic action of insulin on muscle protein metabolism. Experiments will monitor the steady-state distribution of 3H-lysine in candidate precursor pools of cultured embryonic chick skeletal muscle. Peptidyl-tRNA will be isolated from total and myosin-synthesizing polyribosomes and its lysine specific activity compared with those of tRNA lysine, intracellular free lysine and extracellular lysine. Rates of total protein synthesis, myosin synthesis, lysine transport, protein degradation and lysine reutilization will be determined from flux studies in which radiolysine is administered extracellularly or enters muscle pools from prelabeled protein. Measurements will also be made to assess the degree of tRNA acylation and the distribution of lysyl-tRNA isoaccepting species in the total cellular tRNA and in tRNA from total and myosin-synthesizing polyribosomes. This systematic analysis of precursor-product relationships will be followed by experiments to define changes in amino acid pools and protein metabolism caused by insulin. This research will provide an understanding of the relationship between muscle amino acid pools and protein turnover which will allow proper interpretation of isotope data. The research will also permit quantitative conclusions regarding the role of insulin in controlling the rates of muscle protein synthesis and degradation and in regulating the availability of amino acid substrates from both extracellular and intracellular sources.